Alternative splicing of messenger RNA is a common event in humans. A variety of processes contribute to alternative splicing, including use of alternative 5' untranslated regions and initiation codons, polyadenylation signals, cryptic splice sites, overlapping exons, and cassette exons that may or may not be included in a message. In combination, these variations can produce an enormous number of different signals. Understanding the complexity of this transcriptome is a necessary step beyond the Human Genome Project and is the first item in the NHGRI's Grand Challenge. Current tools to identify and quantify splice variants are inadequate. This research project will develop and refine a method for identifying all known splice variants in a genome. This assay will be rapid, quantitative, multiplexed and amenable to high throughput. There is the potential to translate this approach to an array-based platform or to a next- generation sequencing platform to simultaneously study the complete expression patterns for all genes. Pilot studies demonstrate the ability of the oligonucleotide ligation-based assay to form a representation of each alternative transcript. These representations can then be identified and quantified by electrophoresis or other methods. Differences in alternative splicing will be assayed in pairs of diseased and normal tissues. This novel, technology-based project has the potential to produce dramatic advances in our understanding of gene expression by providing a tool for characterizing human gene expression variation. PUBLIC HEALTH RELEVANCE: A large part of health is dependent on genetics, and genes control cells through their RNA. This project will develop a new test to measure the different RNAs produced by a cell. This test will allow us to study the regulation of cells and tissues in health and illness, with the goal of modifying the control systems through drugs.